Reagentless and Reusable Biosensors with Tunable Differential Binding  Affinities and Methods of Making

ABSTRACT

The biosensor comprises a modular biorecognition element and a modular flexible arm element. The biorecognition element and the flexible arm element are each labeled with a signaling element. The flexible arm contains an analog of an analyte of interest that binds with the biorecognition element, bringing the two signaling elements in close proximity, which establishes a baseline fluorescence resonance energy transfer (FRET). When an analyte of interest is provided to the biosensor, the analyte will displace the analyte analog, and with it, the signaling module of the modular flexible arm, causing a measurable change in the FRET signal in a analyte concentration dependent manner. The modularity of different portions of the biosensor allows functional flexibility. The biosensor operates without additional development reagents, requiring only the presence of analyte or target for function.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a Division of application Ser. No. 11/094,540 filedon Mar. 25, 2005, the entirety of which is incorporated herein in fullby reference. Ser. No. 11/094,540 is a Non-Prov of Prov (35 USC 119(e))application 60/559,287 filed on Mar. 25, 2004, the entirety of which isincorporated herein in full by reference.

BACKGROUND OF THE INVENTION

Development of robust, sensitive, and reusable sensors is a strongcurrent scientific priority. As such, recognition-based biosensorscapable of specifically detecting chemicals, toxins, and bio-agents intheir environment are of increasing importance. An important goal inbiosensor evolution is production of nanoscale assemblies capable ofcontinuously monitoring concentrations of target species in a simple,reliable manner. This is accomplished by designing sensor components tocarry out analyte recognition and binding while simultaneously producinguseful output signals via an integrated signal transduction system.Optically addressed biosensors of this type often employ fluorescenceresonance energy transfer (FRET) in signal transduction.

FRET has been employed in carefully designed sensing systems forproteins, peptides, nucleic acids and other small molecules, see Medintzet al., Nature Materials 2003 2, 630. It is known in the art that othersensing modalities can be employed in the signal read-out ofrecognition-based biosensors, especially electrochemical modes or enzymerelated systems, see Benson et al., Science 293, 1641-1644 (2001).

Biosensors function by reversibly linking bioreceptor-target analytebinding with closely integrated signal generation. Such sensors caneither continuously monitor analyte concentrations or easily be returnedto baseline read-out values by removal of analyte. Current bioassays onthe market are single use or limited time use. Either they need to bereplaced after each test or within a short time. This increases bothtest costs and the logistical demands for performing the analysis.Fielded biosensors can have complex robotics that handle the reagentstorage and sensor surface replacement.

Sensor systems based on Surface Plasmon Resonance (SPR) can beregenerated, such as the Biacore SPR instrument. This sensor works bymeasuring the change in index of refraction at the sensor surface uponanalyte binding. This works well for large molecules, but requires aharsh regeneration fluid that limits sensor lifetime. In addition, itworks poorly in complex samples where nonspecific deposition to thesurface interferes with the ability to discriminate actual signal.

Other systems can be used numerous times for the detection of smallmolecules, however as the fluorescent analog is displaced off the sensorsurface the sensor is slowly consumed until it no longer functions. Oneexample is the flow immunosensor of U.S. Pat. No. 5,183,740 to Ligler,et al.

Surface acoustic wave sensors with selective membranes exist for thesensitive detection of gas phase molecules. However, similar devicesbuilt for the liquid phase detection are less sensitive and have thesame limitations as SPR.

FRET based assays have previously been described; however they are fluidphase methods that are effective for a single analysis only. This isalso true for tests that depend on fluorescence anisotropy measurements,which are effective solution phase analyses but require the reagents tobe freely moving to monitor a change upon binding.

A definition describing a biosensor has been proposed by IUPAC whichprovides that “a biosensor is a self-contained integrated device whichis capable of providing specific quantitative or semi-quantitativeanalytical information using a biological recognition element(biochemical receptor) which is in direct spatial contact with atransducer element. A biosensor should be clearly distinguished from abioanalytical system which requires additional processing steps, such asreagent addition. Furthermore, a biosensor should be distinguished froma bioprobe which is either disposable after one measurement, i.e. singleuse, or unable to continuously monitor the analyte concentration”.Although biological recognition elements are employed in an extensiverange of analytical formats, in few cases are they integrated intosensing devices and meet all these rigorous criteria.

The functional simplicity afforded by biosensors, allowing autonomousand continuous monitoring of chemical species, promises to make thesedevices useful in chemical process monitoring, pharmaceuticalsscreening, patient point-of-care and environmental testing, publichealth, and in defense-related fields.

Optically addressed molecular biosensors that meet the above criteriahave been developed by Helling a et al., Proc. Natl. Acad. Sci. USA1997, 94, 4366-4371, in which bacterial periplasmic binding proteins(bPBPs) were engineered to allow transduction of binding events toremote fluorescent signal-generating sites within the same protein byallosteric coupling. Recently, this strategy has been extended to allowsurface tethering to unmodified hydrophobic surfaces of dyelabeled-bPBPs by engineering of a self-adhering hydrophobic peptide ontothe protein terminus, see Wada, et al, J.A.C.S. 2003, 52, 16228-16234.

FRET-based fusion protein biosensors that employ different colored greenfluorescent protein (GFP) mutants linked to substrate binding domainsthat report binding events by coupled changes in conformation and energytransfer have also been developed, see Fehr, et al, Proc. Natl. Acad.Sci. USA 2002, 99, 98469851, and Fehr et al, J. Biological Chem. 2003,278, 19127-19133. Both of these biosensor types are only useful for asmall range of analysis targets. In the bPBP-based sensors,intramolecular transduction of binding events to integrated signalingcenters requires highly specialized allosteric receptors. Even thoughcomputationally intensive redesign of the binding sites for recognitionof alternate substrates may be feasible, binding pocket remodeling isunlikely to prove practical in providing sensors useful for monitoring awide range of analytes. In sensors employing GFP fusion proteins, wheredifferences in FRET efficiency between bound- and analyte-free receptorstates result in signal generation, the range of bioreceptors thatundergo obligatory ligand-dependent conformational changes is also verylimited.

A variety of sensitive FRET-based single-measurement bioanalyticalsystems or bioprobes have been developed that detect peptides, proteins,and various small molecules in vivo and in vitro. See Scheller, et al,Biotech. 2001, 12, 35-40; Iqbal, et al, Biosens. Bioelect. 2000, 15,549-578; O'Connell, et al, Anal. Lett. 2001, 34, 1063-1078; and Marvin,et al Proc. Natl. Acad. Sci. USA 1997, 94, 4366-4371. Maxwell, et al, J.Am. Chem. Soc. 2002, 124, 9606-9612, describes a goldnanoparticle-nucleic acid FRET biosensor that utilizes a probe DNA oligoin a ‘molecular beacon’ function to detect other DNA.

Biosensors that utilize FRET are also attractive due to the intrinsicsensitivity of FRET to small changes in donor-acceptor distance andorientation. Medintz, et al, Bioconjugate Chem. 2003, 14, 909-918demonstrated the feasibility of using dye-labeled MBP and dye-labeledβ-CD for FRET-based detection in solution. Rather than being a sensor,that homogenous system functions only in single-measurement bioanalysis.

BRIEF SUMMARY OF THE INVENTION

This invention provides a FRET-based surface-bound biosensor thatovercomes the single-use limitation of previously known homogenousbioanalytical systems. The invention provides for fully reversible,reagentless, self-assembling biosensors. It is an object of the presentinvention to provide a biosensor design that can easily be adapted totarget different analytes. It is another object of the invention toprovide a biosensor that has a modular design for incorporating a widerrange of available receptors as bio-recognition elements. It is afurther object of the invention to provide a biosensor that usesmultifunctional surface-tethered components. It is a further object ofthe invention to provide a biosensor that is regenerable or can operatein a continuous mode. These and other objects are provided by theinvention disclosed below.

The biosensor of the present invention comprises a modularbiorecognition element and a modular flexible arm element that reactswith the biorecognition element. The biorecognition element is tetheredto a substrate and can be a protein, an aptamer, a carbohydrate, DNA orRNA molecule, among others. This biorecognition element is labeled witha fluorescent dye. The modular flexible arm is also tethered to asubstrate, in an orientation that allows it to interact with thebiorecognition element. The flexible arm contains an analog of theanalyte that binds with the biorecognition element. Both elements arelabeled with a fluorescent dye. When the elements bind, the dyes arebrought in close proximity, which establishes a baseline fluorescenceresonance energy transfer (FRET). When an analyte of interest isprovided to the biosensor, the analyte will displace the analyte analog.The displacement of the analyte analog by the analyte will cause achange in the FRET signal in a concentration dependent manner. Thesensor of this invention can be regenerated and returned to baselinelevels by washing away analyte. A complex set of interactions exists onthe sensing surface that contributes to overall sensor behavior andlargely determines sensor dynamic range. This modular biosensor approachprovides a way to assemble a wide range of useful biosensors that areregenerable, can be reused numerous times, or operated continuously andindependently. Facile control of binding constants and sensing range isa built-in capability. The modularity of different portions of thebiosensor allows functional flexibility. The biosensor is adaptable tomeasure many different analytes or targets. The biosensor operateswithout additional development reagents, requiring only the presence ofanalyte or target for function.

The recognition-based sensors of this invention are capable ofspecifically detecting chemicals, toxins, and bio-agents in theirenvironment. These sensors employ a multifunctional molecular assemblyin which a surface-immobilized biorecognition entity can reversibly bindto a surface-tethered entity immobilized on the same or suitably closelysituated surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of the modular biosensor.

FIG. 2 depicts the structure of a modular arm of the MBP biosensor.

FIG. 3 shows the structure of the dye-labeled TNB DNA arm.

FIG. 4 a shows a schematic of the MBP biosensor in its resting state

FIG. 4 b shows a schematic of the MPB biosensor in the presence of ananalyte

FIG. 5 a shows a schematic of the TNT sensor in its resting state

FIG. 5 b shows a schematic of the TNT sensor in the presence of analyte

FIG. 6 is a series of graphs depicting the change in fluorescence inrelation to analyte concentration.

FIG. 7 is a graph depicting the change in fluorescence in relation toanalyte concentration, demonstrating the effect of controlling bindingproperties when the flexible arm is stiffened with DNA.

FIG. 8 is a series of graphs depicting the change in fluorescents inrelation to analyte concentration.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 depicts a schematic of a modular biosensor of the presentinvention. The biorecognition element (10) and the modular arm element(20) are tethered to a surface (30) by specific oriented surfaceattachment module (40). The surface attachment modules (40) includes,but are not limited to, biotin, avidin, antibody, reactive thiol,reactive amine, non-reversible enzyme substrate, protein A, protein G,protein L, DS-DNA, and PNA. Methods to accomplish surface attachmentinclude, but are not limited to, biotin-avidin chemistry, metal-affinitycoordination, thiol bonding, hydrophobic interactions, and DNA-directedimmobilization. Other means known in the art can be utilized. Thesurface (30) can be a solid planar macromaterial or spherical or othershaped material, or a microscopic planar, spherical or other shapedmicro- or nanomaterial, a nanocrystalline or modified nanocrystallinematerial, or a molecular or biomolecular assembly composed of protein,DNA, RNA, PNA, morpholino DNA, or other biomolecule and theirderivatives, molecularly templated materials, naturally occurringpolymers, minerals, and any similar base material. Those skilled in theart would understand that the biosensor of the present invention couldbe made without the use of a surface (30) by tethering the modular armelement (20) directly to the biorecognition element (10). Thebiorecognition element (10) allows for specific oriented surfaceattachment (40), and contains a biorecognition module (50) that is sitespecifically labeled with a signaling module (60). The types ofbiorecognition module (50) includes, but is not limited to, proteins,such as enzymes, receptors, bPBPs, antibody fragments, and peptides),aptamers, carbohydrates, DNA, PNA, and RNA. The modular arm (20) allowsfor specific oriented surface attachment (40), and comprises a flexiblearm (70) and a recognition module (80), and has been site-specificallylabeled with a signaling module (60). The flexible arm (70) is comprisedof flexible moieties, including, but not limited to, SS-DNA, DS-DNA,combinations of SS-DNA and DS-DNA, thiolated DNA, RNA, thiolated RNA,linear homopolymers, linear copolymers, block copolymers, PNA, αpeptides, β peptides, protein, polymer, or oligosaccharide. Thesignaling module (60) includes, but is not limited to, fluorescent dyes,quenchers, electrochemically active groups, quantum dots and enzymes.The recognition module (80) includes, but is not limited to, antigens,epitopes, analytes, substrates, proteins, peptides, toxins, sugars,biological agents, and analogs of said. The recognition module (80) isattached to the distal end of the flexible arm (70). The binding of therecognition module (80) with the biorecognition module (50) provides abiosensor that is in a ground state by bringing both signaling modules(60) into close proximity to each other. This close proximityestablishes a baseline fluorescence resonancy energy transfer (FRET).Analyte added to the biosensor will competitively displace therecognition module (80). Signal transduction of the FRET is sensitive tothe displacement of the recognition module (80). Binding affinitycontrol can be achieved by, but is not limited to, stiffening of theflexible arm (70), using different affinity biorecognition elements, orby temperature.

It would be understood by those skilled in the art that the biosensor ofthis invention could be easily altered to detect a wide range ofanalytes of interest. Disclosed are the components and architecture of abiosensor as well as a method of preparing and assembling a reagentlessand reusable biosensor with tunable differential analyte bindingproperties. This sensor consists of at least two macromolecular entitiesthat are able to interact with each other for sensing. Both entities canbe tethered to a surface.

FIG. 4 a depicts a schematic view of a biosensor for maltose in itsresting state. The modular arm element (20) is bound to thebiorecognition element, maltose binding protein (MBP), (10) by therecognition module (80), an analyte analog, β-cyclodextrin (β-CD), ofthe analyte of interest, maltose. The signaling elements (60) of thebiorecognition element (10) and the modular arm element (20) are inclose proximity, establishing a baseline FRET. It is understood by thoseskilled in the art that the close proximity for establishing FRETgenerally occurs in the range of about 1 to 10 nanometers.

FIG. 4 b depicts the schematic view of the biosensor after theintroduction of analyte, maltose, (110) to the biosensor. Therecognition element, β-CD, (80) has been displaced by the analyte (110),causing the flexible arm module (20) to move with respect to thebiorecognition element. This causes the signaling element (60) of thebiorecognition element, MPB, (10) and the signaling element (60) of themodular arm element to move with respect to each other, causing ameasurable change in FRET. A detector would provide the means forproviding the excitation light and detection means for measuring theFRET levels. The biosensor (100) can be washed to remove the analyte(110), regenerating the biosensor to the resting state of FIG. 4 a.

FIG. 5 a depicts the schematic view of the TNT biosensor in its restingstate. The modular arm element (20) is bound to the biorecognitionelement, anti-TNT-scFv fragment (α-TNB), (10) by the recognition module(80), an analyte analog, (TNB), of the analyte of interest (110), TNT.The signaling elements (60) of the biorecognition element (10) and themodular arm element (20) are in close proximity, establishing a baselineFRET.

FIG. 5 b depicts the schematic view of the TNT sensor after theintroduction of analyte, TNT (110) to the biosensor. The recognitionelement, α-TNB, (80) has been displaced by the analyte (110), causingthe flexible arm module (20) to move with respect to the biorecognitionelement, α-TNB, (10). This causes the signaling element (60) of thebiorecognition element, α-TNB, (10) and the signaling element (60) ofthe modular arm element to move with respect to each other, causing ameasurable change in FRET. A detector would provide the means forproviding the excitation light and detection means for measuring theFRET levels. The biosensor (100) can be washed to remove the analyte(110), regenerating the biosensor to the resting state of FIG. 4 a.

FIG. 7 shows the effect of adding a modulator to the sensor that altersits binding characteristics. The sensor was self-assembled with Modulararm P1 tethered using oligo Bio3 and MBP95C QSY7 attached with Bio-XNTA. The resulting titration against maltose is show in the top graph A.A variant of the sensor described above with the addition of modulatorDNA is depicted in bottom graph B. Useful sensing range is defined asthe range from 10-90% fractional saturation.

In one embodiment of the invention, the biorecognition element consistsof a specific protein, i.e. maltose binding protein (MBP) that has beendye-labeled. The modular arm element consists of an analyte analog (i.e.beta cyclodextrin), a signaling module (i.e. a cyanine dye), a flexiblearm module (i.e. synthetic oligomeric DNA), and an immobilization module(i.e. biotin). The component modules of the modular arm element arechemically linked to each other in a linear fashion using syntheticchemical methods. In the sensor, the biorecognition module and themodular arm element recognize and bind with each other. Specifically,the protein saccharide binding site binds the cyclodextrin, and as thetwo incorporated dyes in the assembled sensor are in close proximity toeach other, fluorescence resonance energy transfer (FRET) takes placeand can be measured. Upon contact of the assembled sensor with targetanalyte, e.g. maltose or other structurally related sugar, displacementor replacement of the cyclodextrin from the dissacharide binding site ofMBP occurs, causing the two incorporated dyes to move in space relativeto one another, which results in a change in FRET that can be monitored.

Real or apparent binding constants or other relevant physical properties(linked to sensor sensitivity and/or dynamic range and/or chemical orthermal stability) of the biosensor can be altered by hybridizing DNA ofdifferent compositions and lengths to the flexible arm module, yieldingcontrol over sensing kinetics and thermodynamics. Additionally, variantsof MBP with different intrinsic sugar binding properties can besubstituted at will as the biorecognition element of the sensor, whichalso allows adjustment and control of sensor performance.

Because the biorecognition module and modular arm module are tethered toa surface, the target analyte can repeatedly be washed away and thesensor regenerated to a resting state ready for another sensing event.The sensor can also function as a completely independent, continuouslyfunctional monitor for an analyte in an environment where theconcentration of the analyte can change over time, such as in a streamof flowing liquid, where a detector, i.e., a supply of exciting lightand detector of optical signals are present. Furthermore, given thetethered design, protein receptors and analogs that may not normallybind together in solution can be forced to interact cooperatively forsensing events.

The sensing assembly is easily immobilized and assembled on microtiterwell surfaces, takes advantage of biological specificity, links targetbinding with signal generation, and functions in a reversible andregenerable manner. Controlled immobilization and orientation ofproteins while maintaining their relevant activity on a solid surfaceremains challenging. To accomplish this, previous biosensor assemblieshave been described that rely on hydrogel encapsulation of sensorcomponents. The method of the present invention uses an approach thatrelies on self-assembly of components upon a protein-passivated surface.The degree of structural and functional control afforded by thisapproach makes this general assembly method ideal for assembling avariety of receptors for sensing wide range of targets. Beginning withthe concept of surface-tethering all sensor components, several keydesign features were considered. In order to immobilize and organize therequired molecular elements, reliance on readily accessible and robustavidin-biotin assembly methodology was desirable, so the sensor wasassembled on NA-coated microtiter plate wells and monitored with afluorescent microtiter plate reader, technology readily accessible oralready present in many biological testing laboratories.

Methods utilized for surface confinement of both bioreceptor and analyteanalog, maltose binding protein and β-CD in this case, needed to providesufficient freedom of motion for displacement of the analyte analog bythe target analyte during sensor operation. To accomplish this, amultifunctional modular sensor arm was conceived that would bestructurally flexible, yet incorporate the analyte analog, FRET donordye, and surface-tethering element in a single chemically linked unit. Aternary B-CD-Cy3.5-DNA oligonucleotide tether arm that could beimmobilized by DNA directed immobilization (DDI), yet easily made usingcommercially obtained materials, was selected to fill this role.Complementary base pairing specificity in DDI allowed a high degree ofspatial control in sensor self-assembly and placed the extended flexibletether arm onto the NA surface in an oriented fashion in close contactwith the protein receptor. Similarly, oriented immobilization ofHis-tagged MBP bioreceptor variants was assured by using engineeredversions of the protein that allowed single-point attachment to the NAsurface via biotin-X-NTA. As oligohistidine sequences are incorporatedinto many cloned and expressed proteins for metal affinity purification,many types of bioreceptors should be able to function in similarlyconstructed sensors after being labeled with an appropriatesite-specific quencher dye.

Means for potentially modulating sensor performance were also consideredand incorporated in the system. Sensors can easily accommodate‘switching in’ of mutant receptor proteins as well as changes in thelength or composition of DNA tethering elements to effect alteration ofsensor performance.

There are several useful features of the present sensor design. Thesensor can function with either of the two FRET dyes acting as energydonor or acceptor. Although only two dye pairs were used in the currentwork, the numerous FRET dye pairs available can provide the basis forthe construction of multiple sensor versions, including an array or‘multiplex’ sensing surfaces or devices. The present prototype sensorcould be regenerated and re-titrated with maltose several times,yielding essentially the same binding constant with each subsequenttitration. In its present configuration, 8 to 10 uses were possiblebefore useful changes in fluorescence were eliminated, perhaps due tocumulative FRET dye photobleaching, MBP denaturation, or gradualdissociation of sensor components from the polystyrene surface duringwashing. However, the fact that the sensor assembly could beregenerated, re-used and yield essentially the same result is veryencouraging.

Sensors based on this invention can be preassembled, dried, shipped, andreconstituted for use in the field. One advantage is the relativelysmall loss sensor performance due to tethering of all components to asurface. The effective concentrations of the surfaced-confined elements,present in approximately equimolar amounts, are undoubtedly very large,yet relatively low maltose concentrations were able to activate thesensor. The significant broadening of the binding curve for maltose,relative to the binding curve obtained for the components free insolution, indicates that a heterogeneous set of interactions betweensensor components takes place on the surface. A subset of surfacecomponents have relatively optimal binding interactions between tetheredβ-CD and MBP so that they respond only to high concentrations ofmaltose. This subset is useful for sensing soluble sugar at highconcentration, but is unresponsive to maltose at lower concentrations.Another surface-bound component subset forms interactions that are muchless optimal for binding of the pendant β-CD of the DNA-dye-cyclicpolysaccharide tethered arm within the MBP sugar binding site. In thiscase, maltose competes effectively for binding when present at muchlower concentrations. Paradoxically, rather than being a liability forsensing, the effect of this surface heterogeneity is to expand theeffective dynamic range of the system. In fact, this unexpectedlycomplex behavior functions to ameliorate the limited dynamic range thatis a fundamental problem in most recognition-based sensing systems.Furthermore, greater control of sensor dynamic range can be accomplishedby formation of even more complex surfaces, such as demonstrated here byuse of alternate DNA tether arms with hybridized modulator DNA.

Progressive changes of the shape of the analyte binding curve over arelatively small temperature range above ambient (25-40° C.), leading toan endpoint at the upper temperature where sensor response approachesideal apparent two-state binding behavior suggests “melting away” ofmost or all of the low-energy surface interactions contributing tosurface heterogeneity. Initial heterogeneity of the complex sensorsurface leading to the observed behavior could result from a widevariety of molecular interactions. Inter- or intramolecular DNA,DNA-protein (with MBP and/or neutravidin), and/or interactions of any orall of the components with the substrate polystyrene surface could playa role. Most likely, a complex synergistic interplay among the sensorelements due to the above interactions leads to the observed behavior.Predictable control and optimization of sensor surface behavior is agoal for future efforts, in which additional systematic alterations ofsensor component properties (such as very careful design of DNAcomponents to eliminate possible low-melting intramolecular hairpinformation) needs to be carried out. Nonetheless, even in the absence ofextensive optimization of the materials employed, the sensor surfaces wehave constructed are able to function as sensors, and functionrepeatedly, which suggests that the overall assembly strategy employedis quite robust.

The modulation of sensor behavior achieved by changing the structure andcomposition of the synthetic DNA used in the modular tether armdemonstrates a way of fine tuning the sensors of the present. Whilerecently reported work demonstrated irreversible off-to-on switching byannealing of complementary DNA to an engineered enzyme-containingassembly, the present sensor design is the first to make use of DNA-DNAinteractions to modulate the binding properties of a fully reversiblebiosensor assembly. When DNA is used for tethering and modulationpurposes in similar designs, secondary structure considerations must becarefully examined. Furthermore, although examples disclosed have usedDNA for the tethering and modulation functions of the biosensor, a widerange of other materials are feasible for sensor arm construction,including PNAs, peptides, and other linear oligomeric or polymericmaterials. Similarly, non-protein receptors such as DNA or RNA aptamers,templated surfaces, or other types of molecular receptors can functionas recognition elements. Although quenching of organic dyes forbiosensor signal generation was used in the examples herein, use ofoptical components such as quantum dot energy donors, or altogetherdifferent read-out modalities, such as enzymatic or electrochemicalmonitoring, are also possible.

The foregoing example exemplifies only one biosensor assembly that canbe prepared using the architecture, components and methodology describedin the invention. Using different components, but using the same orsimilar basic architecture and assembly methods, a very large number ofsensors, each having the novel features described, may be prepared.

Having described the invention, the following example is given toillustrate specific applications of the invention, including the bestmode now known to perform the invention. These specific examples are notintended to limit the scope of the invention described in thisapplication.

EXAMPLE 1 Biosensor for Maltose

Maltose Binding Protein. In this assembly, the bio-recognition elementsemployed are engineered variants of E. coli maltose binding protein(MBP). MBP, a well-characterized member of the bPBP superfamily, hasbeen used extensively for prototyping purposes in the previouslydescribed biosensors, as well as in redox- and quantum dot-based hybridFRET biosensors. Binding of maltose to MBP is accompanied by aconformational change; however, the FRET-based biosensor does not dependon a change in conformation for signal generation.

As illustrated in FIG. 4 a, Quencher-dye (60) labeled biotinylated E.coli maltose binding protein (MBP) (10) bound in a specific orientationto a NeutrAvidin-coated surface (30) is employed as a biorecognitionelement (10). To complete sensor formation, a flexible arm module (20)comprised of a flexible biotinylated DNA oligonucleotides (115), afluorescence resonance energy transfer (FRET) donor dye (60), and adistal β-cyclodextrin (β-CD) analyte analog (80) is bound in anequimolar amount to the same surface by means of DNA directedimmobilization. After self-assembly, a baseline level of FRET quenchingis observed due to specific interaction between the β-CD of the flexibletether arm and the sugar binding site of MBP, which brings the 2 dyesinto close proximity. Addition of the target analyte, maltose, displacesthe linked β-CD-dye of the DNA-based tether arm, and a concentrationdependent change in FRET results. Biosensor sensitivity and dynamicrange can be controlled by either using MBP variants having differentbinding constants or by binding of modulator DNA oligonucleotides thatare complementary to the flexible DNA tether

The biosensor is self-assembled on a NeutrAvidin™ (NA)-coated surface,and the basic sensing unit consists of an MBP bio-recognition elementthat binds the cyclic sugar β-cyclodextrin (β-CD) that is co-immobilizedon the same surface as part of a multifunctional modular “tether arm.”The distal β-CD element of this modular tether arm is linked to anintegrated dye molecule that functions in signal generation. The targetanalog and signal transduction functionalities (β-CD and dye) in thesame module are anchored to the sensor surface by hybridized DNAinteractions via DNA-directed immobilization (DDI) utilizing modifiedoligonucleotides. Following biosensor assembly, an appropriate sugaranalyte (e.g. maltose) can compete with β-CD for binding at the MBPbinding site. Movement of the β-CD analyte-analog into or out of theprotein binding site is coupled with obligatory movement of theintegrated signaling dye, and this spatial displacement forms the basisfor quantitative reporting of analyte sugar concentration by changes inFRET. The present biosensor, which is composed of molecular componentsthat exist in direct spatial contact, and that is functional in aquantitative, continuous, and reversible manner that requires noadditional or secondary reagents meets the rigorous criteria that definea biosensor.

MBP95C, MBP80C and MBP-AT (containing the specific AviTag recognitionsequence for in vivo biotinylation) were engineered and expressed asdescribed below. The DNA coding sequence for MBP protein (Mr˜44 kD) iscontained on a standard multicopy plasmid vector containing theampicillin resistance gene and was expressed. This MBP gene sequence wasengineered to express a C-terminal 5 histidine sequence along withaspartate 95 or threonine 80 changed to cysteine (MBP95C, MBP80C) usingstandard gene assembly and cloning techniques. To create an MBP thatexpresses the AviTag specific biotinylation sequence, a MBP mutantplasmid was engineered with codons changed to those recognized by XhoIand HindIII 27-30 and 15-18 bps, respectively, from the C-terminalpenta-histidine coding sequence. The plasmid was then digested with XhoIand HindIII and oligonucleotides for a modified AviTag sequence ligatedinto these sites which coded for the sequence: AGLGGLNDIFEAQKIEWHE. Invivo, biotin is covalently attached to the lysine, K, residue in thissequence. Transformants were screened and sequenced for correctintegration. This MBP-AT (AviTag) plasmid was co-transformed into E.coli strain AVB99 along with pACYC184 (Avidity, Denver Colo.), whichexpresses the biotin holoenzyme synthetase BirA. Cells were grownovernight in 100 ug/mL ampicillin/10 ug/mL chloramphenical media andinduced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and 50 μMd-biotin. MBP-AT was purified and biotinylated MBP-AT was furtherpurified using the ImmunoPure Immobilized Monomeric Avidin Kit (Pierce,Rockford Ill.). The MBP sequence (SEQ ID NO:1) with locations of 80C and95C highlighted is below. Position of codons changed to XhoI and HindIIIalso shown.

SEQ. ID NO: 1 Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn GlyAsp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp ThrGly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro GlnVal Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg PheGly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile                                     *80* Thr Pro Asp Lys Ala Phe GlnAsp Lys Leu Tyr Pro Phe Thr Trp Asp                                          *95* Ala Val Arg Tyr Asn GlyLys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn LysAsp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp LysGlu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro TyrPhe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu AsnGly Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala GlyLeu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr AspTyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile AsnGly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val ThrVal Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu SerAla Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu GluAsn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro LeuGly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg IleAla Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro GlnMet Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser GlyArg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Lys Leu            /XhoI /       / HindIII/ Thr Lys Gly Ser His His His His His

Mutant proteins were specifically labeled on cysteine residues usingmaleimide activated Cy3 dye (extinction coefficient 150,000 M⁻¹cm,quantum yield >0.15, Amersham Biosciences, Piscataway N.J.) or QSY 7 dye(extinction coefficient 90,000 M⁻¹cm⁻¹, Molecular Probes, Eugene Oreg.).

β-CD-Cy3.5-P1 DNA Synthesis and Mass Spectral Analysis: BisfunctionalNHS-ester activated Cy3.5 dye (extinction coefficient 150,000 M⁻¹cm⁻¹,quantum yield >0.15, Amersham Biosciences) solubilized in 0.136M Natetraborate buffer pH 8.5 was added to a vial containing ˜5 nanomolesamino-functionalized P1 DNA. After reacting for 20 min at roomtemperature in the dark, a 100-fold excess ofmonoamino-beta-cyclodextrin was added. After 4 hrs, 200 μL of HE buffer(10 mM HEPES, 1 mM EDTA, pH 7.0) was added and the product precipitatedwith 25 μL—3M NaCl and 650 uL cold ETOH at −20° C. The precipitatecollected by centrifugation was washed 4 times with ETOH and dried in avacuum centrifuge. Dried pellets were solubilized in HE buffer andsynthetic products separated from underivatized dye on 12% acrylamidegels by electrophoresis. Product band(s) were excised and theβ-CD-dye-DNA adduct (abbreviated as P1) eluted by passive diffusion intoHE buffer. Adduct(s) were concentrated/desalted using an oligonucleotidepurification cartridge, OPC. Pure products were subjected to massspectral analysis. Samples were de-salted and removed from non-volatilebuffer(s) by solid phase extraction using C18 zip tips. Samples werebound to the tip and washed 3× with high purity water, 0.1%trifluoroacetic acid (TFA). Samples were subsequently eluted with 10 μLof 75% acetonitrile (ACN), 0.1% TFA, added to 90 μL of MALDI matrix (35mg/ml 3-hydroxypicolinic acid in 30% ACN, 10 uL of 50 mg/ml ammoniumcitrate). One μL of each solution was deposited onto the MALDI plate.Samples were analyzed by MALDI-TOF mass spectrometry using a Voyager-DEPRO. Using the negative ion and linear TOF modes, signal was detectedfor the ternary product consisting of cyclodextrin-Cy3.5 dye-P1 DNA atm/z 7068. The mass calculated for the cyclodextrin-Cy3.5 dye-P1 amineDNA complex (P1) was 7073 amu. The synthetic product consisting of P1DNA-Cy3.5 dye (without appended β-CD) was similarly isolated andidentified.

Sensor Assembly: Reacti-Bind Neutravidin (NA) Coated Plates or opaquewhite microtiter plates coated with NA were prepared. For eachmicrotiter well, 7-10 picomoles of P1 (SEQ ID NO. 2) were allowed tohybridize to the same amount of complementary biotinylated attachmentDNA (Bio1 (SEQ ID NO. 3), 2 (SEQ ID NO. 4), 3 (SEQ ID NO. 5)) inTris-EDTA buffer pH 8 (TE). Modulator DNA Mod1 (SEQ ID NO. 6), Mod2 SEQID NO. 7) was added where indicated in 2-fold concentration. The DNAcontaining solutions were heated to 80° C. for 5 minutes and then cooledslowly to room temperature to preclude any possible secondary structureformation. This solution was then diluted into 50 μL PBS and added toeach NA microtiter well for 1 hour of binding at RT. Wells were washedwith 200 μL PBS and 10 picomoles of biotinylated-MBP-AT95C-Cy3 added toeach well in 50 μL PBS where indicated. Alternatively, 50-80 picomolesof MBP95C-QSY7 or MBP80C-QSY7 was preincubated with an equimolar amountof biotin-X nitrilotriacetic acid tripotassium salt (Biotin-X NTA,Biotium, Hayward Calif.) for 2 hrs at room temperature and added in 50μL PBS to wells containing P1. The final, mixed sensor was allowed tobind and self assemble for 1 hr at room temperature, washed with 200 μLPBS and equilibrated in 50 μL PBS for 15 min before addition of sugarfor testing. Sensors were regenerated by washing with 20 volumes ofbuffer and allowing the sensor to re-equilibrate for 20 min to 1 hr in50 μL PBS. The table below lists oligonucleotides sequences referenced.In the table, sequence P1 is SEQ ID NO:2, Sequence Bio1 is SEQ ID NO:3,Sequence Bio2 is SEQ ID NO:4, Sequence Bio3 is SEQ ID NO:5, SequenceMod1 is SEQ ID NO:6, and Sequence Mod2 is SEQ ID NO:7.

# of TM SEQ ID NO: Name nucleotides Sequence 5′-3′ ° C. SEQ ID P1 16NH2-C₆-ATACCGTTCGCGCCCG 61.8 NO: 2 SEQ ID Bio1 24Bt-C₁₅-ACTGACTGCGGGCGCGAACGGTAT 69.7 NO: 3 SEQ ID Bio2 34Bt-C₁₅-ACTGACTGGAATCTGAATCGGGCGCGAA 71.9 NO: 4 CGGTAT SEQ ID Bio3 44Bt-C₁₅- 74.0 NO: 5 ACTGACTGTACGAGTTGAGAATCTGAATCGGGC GCGAACGGTAT SEQ IDMod1 15 GATTCTCAACTCGTA 48.0 NO: 6 SEQ ID Mod2 20 ATTCAGATTCTCAACTCGTA54.3 NO: 7

Fluorometry and Titration: Fluorometric analysis of microtiter wellplates was performed on a Safire Dual Monochromator MultifunctionMicrotiter Plate Reader. For the Cy3-Cy3.5 fluorophores, samples wereexcited at 520 nm and emission collected at 605 nm. Excitation at 550 nmand 600 nm emission were used for the QSY 7-Cy3.5 pair. For titrations,5 μL of each appropriate sugar solution was added and mixed at RT for 5min as described. Background fluorescence was subtracted and datanormalized for volumetric changes. Data was transformed using a Hill4-parameter plot and approximate binding constants, K_(app), estimatedin these conditions of multiple equilibria along with useful sensingranges using SigmaPlot. For all titrations, the Hill coefficientapproached 1, indicating a 1:1 maltose/MBP interaction.

Sensor Self-Assembly and Testing: Organized self-assembly of both thedye-labeled MBP receptor and extended modular arm bearing the β-CDanalyte analog and reporter dye occurs on NA coated microtiter plates.It is possible to assemble the sensor components either by mixingequimolar amounts of the dye-labeled biotin-functionalized MBP andβ-CD-Cy3.5-P1DNA/Bio1, 2, 3 modular tether arm in solution beforeattachment to the NA surface or by allowing the separate components tobind separately and sequentially. If the separate components bindseparately and sequentially, empirically determine the amount of firstcomponent bound to the surface to leave enough remaining accessiblebiotin-binding sites for filling in with the second sensor component.

The NA biotin binding sites on the surface are ˜50% occupied by abiotin-X-NTA linker coordinated to MBP through a C-terminalpentahistidine extension. Depending on the sensor variation, theremainder of the biotin binding sites are occupied by single-strandedDNA oligomers of varying lengths (termed Bio1, Bio2, or Bio3 of 24, 34,44 bases in length, respectively). Each surface-bound oligo being linkedto the surface through a flexible 5′ hydrophilic 15-atom biotinylatedTEG (triethethyleneglycol) linker on one end and hybridized toβ-CD-Cy3.5-P1DNA on the other. Thus, in the completely assembled sensor,the β-CD-FRET dye modules critical for sensing are held on the surfacein close molecular proximity with bound MBP to form active sensingcomplexes.

On the fully assembled sensor surface, an equilibrium is established inwhich immobilized MBP is positioned to bind the pendant β-CD of themodular arm element within its sugar binding site. With this bindingevent, the FRET dye pair is simultaneously brought into position for abaseline level of fluorescence quenching to occur. Due to the relativelysmall size of the MBP molecule and favorable Forster R0 values for thedyes used, energy transfer between a fluorophore located at the bindingsite of MBP and a dye label placed virtually anywhere on the protein ishighly efficient.

Native MBP binds both the disaccharide maltose (KD of 0.9 μM), and β-CD(KD of 1.8 μM) with similar affinities. In the biosensor, signalgeneration is designed to occur when added maltose competitivelydisplaces β-CD from the MBP sugar binding site. An increasing degree ofoccupancy of the binding site by an increasing concentration of maltose,concomitant with decreasing binding site occupancy of the tethered β-CD,is coupled quantitatively with changes in FRET occurring betweenincorporated system dyes, forming the basis for sensor function. Thesensor assembly is designed to be re-set to a baseline quenching stateby simply removing maltose by washing/dilution, so that quantitativesensing can be carried out repeatedly. Alternatively, when assembled ina properly configured flow cell or fixed within a bioreactor, aderivative of this molecular sensor assembly may be able to continuouslyand reversibly monitor fluctuations in maltose concentration.

Below is the structure of the three part tetherable modular arm and therelevant absorption.

In this configuration, MBP was labeled at either of two engineeredresidues (80C or 95C) by thiol-reactive QSY 7, a dark (non-emitting)quencher dye. β-CD-Cy3.5-P1 (SEQ ID NO: 2) DNA was bound to the surfacevia the 34 nucleotide Bio2 (SEQ ID NO: 4) oligo and MBP95C-QSY7 wassurface localized via biotin-X-NTA. In the initial state of the sensor(no maltose), proximity of the Cy3.5 donor dye of the tethered arm toMBP-bound QSY 7 acceptor dye resulted in partial quenching of Cy3.5fluorescence. Emission of the Cy3.5 increased upon addition of maltose,strongly suggesting that competition for the MBP sugar binding siteoccurs between the soluble disaccharide and the β-CD moiety of theβ-CD-Cy3.5-P1DNA modular arm. A change in average FRET quenchingefficiency occurred due to a net difference in dye-dyedistance/orientation in the system. The binding curve obtained was asmooth function of maltose concentration, but was relatively broad inthe concentration dimension compared with titrations using solublecomponents (see below) suggesting that physical constraints related totethering lead to inhomogeneity in the binding and signal generatingapparatus. Nonetheless, the assembly performed effectively in sensingmaltose.

After saturation with maltose, the sugar could be removed and the sensorreset to its baseline quenching value by rinsing the surface withbuffer. Titration could then be carried out with essentially identicalresults, followed by repeated rounds of regeneration and sensing. TheK_(app) derived for maltose binding to this sensor was 6.7±0.7 μM.Control experiments were performed in which the β-CD-Cy3.5-DNA modularsensing arm was replaced with a partial sensing arm consisting ofCy3.5-P1DNA without the β-CD target analog recognition element.Titration of this control sensor assembly with maltose gave essentiallyno change in emission spectra or fluorescence intensity, demonstratingthat function of the sensor depended on recognition of the β-CD moietyby MBP. Replacement of MBP with a non-functional protein (apo-myoglobinbearing a C-terminal hexahistine) also resulted in no change in baselineemission with added maltose. The fluorescence does drop withregeneration, suggesting an upper limit to the number of regenerationseven though essentially the same binding constant is derived withsubsequent titrations.

Fluorescence changes during titration with maltose were also monitoredwith the same components present both in solution phase and in a“half-tethered” configuration. Starting with an equilibrium-statesolution of 500 nM MBP95C QSY7 and 50 nM β-CD-Cy3.5-P1DNA, systematicchanges in quenching that occurred due to competitive displacement ofβ-CD by maltose yielded a K_(app) of 1.9±0.7 μM. Under these relativelydilute conditions, chosen to avoid inner filter effects, the β-CDcontaining reagent would be expected to occupy about 5-10% of theavailable binding sites of the MBP95C QSY7 present at equilibrium. The˜5% fluorescence change that occurred at maltose saturation wasconsistent with essentially complete displacement of theβ-CD-Cy3.5-P1DNA. The binding curve for the soluble system was morecompressed on the concentration axis compared to the binding curve ofthe fully tethered sensor, reflecting more ideal titration behavior forcomponents freely diffusing in solution, and the K_(app) value obtainedfrom the binding data are consistent with previous MBP solution phasetitrations.

In another titration, MBP95C QSY7 was present in solution at 40 nM,while β-CD-Cy3.5-P1DNA was tethered to the NA surface via DDI withbiotinylated oligonucleotide Bio2. A K_(app) for maltose binding of6.0±0.6 μM was determined and the shape of the binding curve indicatedretention of the essentially ideal binding behavior observed for thefully soluble system; thus, the cyclic sugar of surface-tetheredβ-CD-Cy3.5-P1DNA appeared to be presented to the soluble proteinreceptor in an essentially homogenous manner, unlike the fully tetheredsensor assembly where geometric constraints leading to surfaceinhomogeneity are likely responsible for the broadened binding curveobserved.

The behavior of the biosensor has only minimal dependence on the lengthof this tethering segment over the ranges investigated. Immobilizationsegments Bio1, Bio2, or Bio3 DNA of 24, 34, or 44 bases in length,respectively, were tested. The table below illustrates the sensorproperties.

Sensor Functional Arm Estimated useful % Fl increase upon Protein¹ ArmAttachment Modulator K_(app) μM sensing range² sensor saturation³Comment MBP95C P1 NA NA  1.9 ± 0.7 350 nM-6 μM   5 Both components insolution MBP95C P1 Bio2 NA  6.0 ± 0.6 2.5 μM-10 μM  6 Only P1 tetheredvia Bio2 MBP95C P1 Bio2 NA  6.7 ± 0.7  1 μM-50 μM 10 P1 tethered viaBio2 & MBP95C tethered via Bio-X NTA MBP95C P1 Bio3 1  4.0 ± 0.6  50nM-650 μM 7 Modulator present MBP95C P1 Bio3 2 11.3 ± 1.9 10 nM-1 mM  9Modulator present MBP95C P1 Bio1 NA  3.0 ± 0.3  1 μM-90 μM 10 MBP95C P1Bio3 NA  4.8 ± 0.3 500 nM-30 μM   12 MBP80C P1 Bio3 NA 575 ± 15 10 μM-5mM  133 MBP80C P1 Bio3 2 404 ± 10 7 μM-5 mM 127 Modulator present MBP80CP1 Bio3 2 375 ± 25 3 μM-3 mM 36 ⁴See below MBP95C/MBP80C P1 Bio3 NA 153± 27 4 μM-8 mM 86 Composite sensor MBP-AT95C P1 Bio3 NA 10.5 ± 1.4   1μM-150 μM 6 Biotinylated MBP-AviTag MBP-AT95C P1 Bio3 2  9.3 ± 1.3 500nM-100 μM  5 Biotinylated MBP-AviTag NA—Not applicable. AT—AviTag. Allreadings are performed at 20-22° C. See Suppl. for oligonucleotidesequences and FIG. 1 for schematic ¹Unless otherwise noted, the proteinis tethered to the neutravidin surface via the 5-histidine sequenceusing Ni-X NTA as in Methods ²This is the estimated concentration rangefor which these sensors can be used usefully and is defined as the rangebetween 10% and 90% saturation.^(ref 15,17) ³Calculated as (Fl_(sat) −Fl₀)/Fl₀ × 100 where Fl_(sat) = fluorescence at saturation and Fl₀ =fluorescence before the addition of sugar ⁴Plate with sensors in wellstitrated 4 times consecutively, air dried and stored for 2 weeks inrefrigerator and then titrated 3 times consecutively

Using an estimated value of 3.4 Å for each base in a fully extendedssDNA linker, these linkers varied from ˜82 to ˜150 Å in potentiallength of the single-stranded segment. Binding constants obtained forthese sensor variations remained in the low micromolar range, varying nomore than ˜2-fold, 6.7±0.7 μM for Bio2 versus 3.0±0.3 μM for Bio1. Thebehavior of the system assembled with Bio3, the longest tetheringelement, was similar to that for Bio1. The shape of the binding curvewas similar in each case, and the net change in fluorescence betweenmaltose-free and maltose-saturated states for each of the threevariations tested was ˜10%, demonstrating that the degree of functionalsurface coverage was not dependent on the ssDNA tether length. Thebehavior of the biosensor has only minimal dependence on the length ofthis tethering segment over the ranges investigated.

Modulation of Binding Affinity and Useful Sensing Range Effective meansto modulate the sensitivity and/or dynamic range of the biosensor weresought. The first way explored for controlling sensor behavior involvedrelatively straightforward substitution of an MBP variant with alteredsugar binding properties. A second variation on this form of controlinvolved formation of a “mixed” composite sensor consisting of two sugarreceptor variants on the same surface having different maltose bindingprofiles. A third level of sensor control involved hybridization of“modulator” DNA strands to the residual single-stranded section of theβ-CD-Cy3.5-P1DNA/Bio3 tether arm in an attempt to alter the extension orstiffness of the tether arm by altering the ratio of ss/ds DNA in thismodular component.

As a first level of control, QSY 7-labeled MBP variant MBP80C havingsignificantly different maltose binding characteristics from QSY7-labeled MBP95C was used as the bioreceptor in order to modulatesensitivity and dynamic range. In this MBP variant, labeling atengineered cysteine-80 with the reporter dye significantly increases itsK_(app). Solution phase titration of Cy3-labeled MBP80C under similarconditions yielded a maltose KD of 70 μM. FIG. 8, in the graph labeledA, shows the binding curve from maltose titration of a sensor consistingof the β-CD-Cy3.5-P1DNA modular arm tethered with Bio3 and assembledwith MBP95C QSY7, while FIG. 8, in the graph labeled B, shows data forthe sensor constructed with MBP80C QSY7. The K_(app)'s and usefulsensing ranges for the different sensor configurations are 4.8±0.03μM/500 nM −30 μM and 575±15 μM/10 μM −5 mM, where useful sensing rangewas estimated from 10 and 90% sensor response to maltose. From theseresults it was clear that facile tuning of sensor sensitivity anddynamic range can be accomplished by altering receptor affinity.Combining receptor variants which have differing binding properties intoa composite biosensor is one approach to creating biosensors withchanged dynamic ranges. FIG. 8, in the graph labeled C, presents theresults from maltose titration of a composite sensor assembled withequimolar proportions of MBP95C QSY 7 and MBP80C QSY7 receptors on thesurface. Using this sensor assembly, there was a significant shift inK_(app) (153±27 μM) and useful sensing range of ˜4 μM-8 mM vs. 4.8±0.3μM and 575±15 μM for the MBP95C QSY 7 and MBP80C QSY7 sensors,respectively. Not surprisingly, because of the significantly differentintrinsic AF values for the two base sensor configurations, compositesensor properties were largely dominated by MBP80C QSY7 binding andsignaling. By more closely matching net AF values of the constituentreceptor components or adjusting ratios of each constituent sensor inthe composite, finer control of sensor function could likely beachieved. Combinatorial screening methods could also be effective inoptimizing composite sensors.

A third means to control sensor performance is modification of sensorproperties by altering the extension or stiffness of the tether arm bychanging the ratio of ss/ds DNA in β-CD-Cy3.5-P1DNA modular componentwas explored. Hybridizing complementary DNA to the tether arm willchange a significant portion of the DNA in the tether arm fromsingle-stranded (ss) to a more rigid double-stranded (ds) DNA form, andthe resultant changes in rigidity of the DNA linker arm might allowcontrol of sensor performance. The β-CD-Cy3.5-P1DNA/Bio3/Modulator DNAtether arm was self-assembled using DNA annealing by slow-cooling thecombined heat denatured components (Temperature for all dsDNA-hybrids inthe sensor was designed to be >65° C.) prior to room temperatureimmobilization on the NA surface along with MBP95C QSY 7. In the“unmodulated” β-CD-Cy3.5-P1DNA/Bio3 arm, only 16 of the available 44bases in the Bio3 oligonucleotide (˜35%) were involved in DNA duplexinteractions. Hybridizing complementary MOD1 or MOD2 DNA to Bio3 in theβ-CD-Cy3.5-P1DNA/Bio3 complex raises this number to 31/44 bases (˜70%)or 36/44 bases (˜82%), respectively. FIG. 8, in the graphs labeled D andE, show binding curves obtained from maltose titration of the MBP95C QSY7 sensor with MOD1 or MOD2 oligonucleotides hybridized to theircomplements within the β-CD-Cy3.5-P1DNA/Bio3 modular assembly. Theshapes of the binding curves for the modulated assemblies differconsiderably from the binding curve of the unmodulated sensor, anddifferent K_(app) values and useful sensing ranges for the modulatedassemblies are observed. Specifically, the binding curves for theoligonucleotide modified sensors are broader and shallower than that ofthe parent sensor, and the useful sensing ranges are correspondinglygreater, ranging from 500 nM-30 μM for the unmodified sensor to 50nM-650 μM for MOD1 and 10 nM-1 mM for MOD2 containing MBP95C QSY 7sensors. Similar experiments utilizing MBP80C QSY7 resulted in smallchanges in K_(app), but in this case the presence of modulator DNA didnot significantly alter the useful sensing range. Changes in the packingof surface components due to the presence of modulator DNA could alterthe binding behavior of MBP. Additionally, MBP is known to be verysensitive to small structural changes even distal from the binding site,which can significantly alter affinity. Clearly, this means offine-tuning sensor properties can be successfully implemented, but atpresent the more direct methods previously described are morepredictable and robust ways to modulate sensor function.

Stability of the immobilized sensor assemblies. Sensors assembled usingMBP80C QSY7 and β-CD-Cy3.5-P1DNA/Bio3/MOD2 components were titrated withmaltose in microtiter plate wells, purged of maltose bywashing/dilution, then dried and stored at 4° C. for 2 weeks todetermine long term stability and regenerability of the sensors.Following rehydration with PBS, successful maltose titration was carriedout three times with regeneration. The K_(app) of 375±25 μM obtainedfrom the sensor attached to the dry-stored plate was identical, withinerror, to that of the initially tested sensor.

Sensor System Bioenergetics and Specificity. Maltose titration behaviorwas examined over a range of temperatures. Increasing concentrations ofmaltose were added to wells of a microtiter plate containing identicalsensor assemblies based on MBP80C QSY 7 and β-CD-Cy3.5-P1DNA/Bio3. Afterequilibrating and reading the plate at 25° C., the plate wasre-equilibrated at 30° C. and the fluorescence read with the same at 35and 40° C. Of particular interest was the systematic change in the shapeof the binding curve with temperature, from a broader binding isothermat 25° C. to a more steeply rising curve at 40° C., suggesting that withincreasing temperature the range of functional configurations of thetethered components becomes smaller, resulting in more ideal bindingbehavior at higher temperature. Sensor function is thus significantlytemperature dependent. However, the temperature dependence of the shapeof the binding curves makes it impossible to derive meaningfulthermodynamic parameters in this complex system of multiple equilibria(e.g. Vant Hoff plots are likely to lack quantitative meaning). It islikely that sensor binding thermodynamics and kinetics would be affectedby changes in other physical parameters such as pH, ionic strength andviscosity.

The ability of these surface-tethered maltose sensors to discriminateamong different types of sugars was investigated by carrying outtitrations with a range of sugars, including D-arabinose, D-galactose,D-glucose, lactose (an epimer of maltose), sucrose and β-CD, withmaltose serving as the comparative standard. The table below

Sugar D-Arabinose β-Cyclodextrin D-Galactose D-Glucose Lactose MaltoseSucrose H2O P1-Bio3-MBP95C-QSY7-Bio-X NTA Type¹ Mono Oligo (7-monomers)Mono Mono Di Di Di NA Oligosaccharide NA α-1,4 NA NA 1,4-α α-1,4 α-1,β-2 NA Linkage glucosidic glucosidic glucosidic glucosidic Δ FL² 100 nM<1% 14% <1% <1% <1% 100% <1% <1% Δ FL² 1 mM <1% 58% <1% <1% <1% 100% <1%<1% P1-Bio3-MOD2-MBP95C-QSY7-Bio-X NTA Type¹ Mono Oligo (7-monomers)Mono Mono Di Di Di NA Oligosaccharide NA α-1,4 NA NA 1,4-α α-1,4 α-1,β-2 NA Linkage glucosidic glucosidic glucosidic glucosidic Δ FL² 100 nM<1% 28% <1% <1% <1%  60% <1% <1% Δ FL² 1 mM <1% 73% <1% <1% <1% 100% <1%<1% ¹Mono, Di and Oligo - saccharide ²Δ FL = Percentage increase in PLintensity with respect to the initial value of the nanosensor, uponaddition of the indicated amount of sugar, the response to 1 mM maltosedefines the 100% comparison response NA—Not applicable

With sensors composed of either β-CD-Cy3.5-P1DNA/Bio3 orβ-CD-Cy3.5-P1DNA/Bio3/MOD2 and MBP95C-QSY 7, the only non-zero responseobtained, excepting maltose was for β-CD. The binding specificity of MBPis fully retained on the sensor surfaces prepared.

Biotinylated MBP-AviTag Sensor Variant: Oriented attachment of thedye-labeled MBP to the NA surface for sensor construction has beenaccomplished in two different ways. The first method uses theheterobifunctional crosslinker Bio-X-NTA (where Bio is biotin, Xrepresents an amino-methoxy spacer, and NTA stands for thenitrilotriacetic acid chelator functionality) to link C-terminallyHis-tagged MPB variants to the surface NA. Another MBP variation,MBPAT-95C, was also employed in sensor assembly. In this protein, anAviTag biotinylation recognition peptide was genetically fused to theC-terminus of the MBP sequence. The AviTag sequence allows specific invivo biotinylation by the biotin holoenzyme synthetase BirA andsubsequent isolation of site-specifically biotinylated proteins directlyfrom E. coli lysates. MBPAT-95C was created to investigate whether MBPbiotinylated in vivo could be used for attachment to the NA surface,thus eliminating the need for use of Bio-X-NTA or other potentiallynon-specific chemical biotinylation steps. Cy3-labeled MBPAT-95C wasused to construct and test a Cy3-Cy3.5 emissive variant, where the Cy3.5dye of β-CD-Cy3.5-P1DNA acts as an emissive

FRET acceptor. The K_(app) for maltose binding of 10.5±1.4 was similarto the value derived from MBP immobilized using Bio-X-NTA. Cy3-labeledMBPAT-95C functions well as the receptor, even though it is tethered tothe surface by the biotinylated lysine of the 19-residue C-terminalAviTag peptide. Use of modulator DNA did not significantly alter sensorfunction. In vivo biotinylation of receptors thus represents anotherfacile means to create components for use in sensor self-assembly.Successful use of this alternate receptor immobilization method impliesthat flexibility exists in the assembly strategy described herein.

EXAMPLE 2 TNT Biosensor

The self-assembled modular sensing strategy can be applied to thedetection of an analyte completely unrelated to maltose, namely,2,4,6-trinitrotoluene (TNT), by altering two modules of the sensorassembly. Using the same sensor platform, a dye-labeled anti-TNTsingle-chain Fv antibody fragment (R-TNTscFv) was substituted for theMBP bioreceptor portion of the sensor and the multifunctional tether armwas modified to contain a dye attached to a modified internal base. TheDNA arm is further modified to terminate with the TNT analogue1,3,5-trinitrobenzene (TNB). FIGS. 5 a and 5 b schematically depict theTNT sensor. The R-TNTscFv portion is allowed to bind the TNB analogue,bringing both dyes into proximity, establishing a baseline level ofFRET, and this complex is then self-assembled on a NA surface. Additionof TNT to the sensor solution results in a concentration-dependentchange in FRET. In this configuration, the sensor retains its analytespecificity yet can still have its dynamic sensing range usefullyadjusted. The sensor can be washed free of analyte and reforms forsubsequent detection events. The demonstration that this sensor can bereadily adapted to a completely different analyte highlights the utilityof its modular construction and its general applicability to a varietyof targets.

Explosive standards including TNT, TNB,hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX),2-amino-4,6-dinitrotoluene (2A-4,6-DNT), and 2,4-dinitrotoluene(2,4-DNT) were obtained from Cerilliant Corp. (Austin, Tex.). ReactibindNeutravidin-coated plates were obtained from Pierce (Rockford, Ill.).

Mutagenesis and Protein Expression. The R-TNTscFv fragment, a derivativeof TNB2 described in Goldman, et al, Journal of Env. Monitor; 2003, 5,380-3, was engineered to express aHISHISHISHISHISHISGLYGLYSERGLYGLYHISHISHISHISHISHIS (SEQ ID NO:10)carboxy terminus, where (His)₆=6-histidine residue sequence. Selectedcysteine mutations were introduced into the DNA coding sequence usingthe Quickchange Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.). For mutagenesis, polyacrylamide gel-purified oligonucleotideswere obtained from Sigma Genosys (Woodlands, Tex.). Transformants wereselected, and correct mutational residue changes were verified by DNAsequencing. α-TNTscFv-Cys mutants were expressed in the E. coli Tunerstrain (Novagen, San Diego Calif.) and purified from the periplasm.α-TNTscFv-Cys mutant samples were labeled with maleimide-activatedAlexaFluor 532 dye (AFF 532: quantum yield 0.8, molar extinctioncoefficient 81 000 M⁻¹ cm⁻¹; Molecular Probes, Eugene Oreg., with themodification of using 100× less of the dithiothreitol reducing agent.Dye-to-protein ratios of ˜1 were obtained. Construction and purificationof (His)₆-appended myoglobin, with a cysteine mutation at position 64,and its subsequent labeling with Cy3-maleimide (Amersham Bioscience,Piscataway, N.J.). ELISAs for determining TNB binding were performed onthe α-TNTscFv-Cys mutants.

Dye-Labeled TNB DNA Arm: FIG. 3 depicts the dye-labeled TNB DNA arm. Theprecursor of the dye-labeled TNB DNA arm, purchased from Qiagen(Alameda, Calif.) was already modified with a teramethylrhodamine(TAMRA) dye-labeled internal T* base (quantum yield 0.7, molarextinction coefficient 91 000 M⁻¹ cm⁻¹) and a 5′-amino modification on aC-6 linker (SEQ. ID. No. 12). The DNA precursor was subsequently reactedwith a 1000-fold molar excess of 2,4,6-trinitrobenzene-sulfonic acid (5%solution, Sigma, St. Louis, Mo.), in 1 mL of 0.136 M sodium tetraboratebuffer pH 8.5 supplemented with 20 μL of 2 M NaOH. The resultingsolution was reacted overnight at room temperature under continuousagitation, then loaded on a Supelclean LC-18 SPE column (Supelco,Bellefonte Pa.), washed with 0.1× borate buffer, and eluted with anincreasing concentration of methanol in borate buffer. The mass of thefinal product (MW ˜5878) was verified on an Applied Biosystems API QSTARPulsar mass spectrometer by positive electrospray ioniza-tion.

Sensor Assembly: For each well of the Reactibind NA-coated plates(binding capacity ˜60 pmol of biotin), 30 pmol of dye-labeled TNB DNAarm was added to 30 pmol of hybridizable flexible DNA linker (SEQ. IDNo. 11), heated to 90° C. for 5 min, and then cooled to room temperatureslowly to preclude secondary structure formation. The DNA meltingtemperature is ˜62° C. Concurrently, 30 pmol of AlexaFluor 532-labeledα-TNTscFv was mixed with 30 pmol of biotin-X nitrilotriacetic acidtripotassium salt precharged with nickel (Bio-X NTA, Biotium, HaywardCalif., Bio-biotin, X-aminomethoxy spacer, NTA-nitrilotriacetic acidchelator. After 2 hours, DNA and protein solutions were mixed together(now equimolar), diluted with phosphate-buffered saline pH 7.4 (PBS) toa final volume equivalent to 50 μL/well and incubated for 4 h at roomtemperature. A 50-μL aliquot of sensor solution was added to each wellof the NA plates and incubated at 4° C. overnight. For regeneration,sensor-coated plates were washed 10× with 200 μL of PBS andreconstituted in 50 μL of PBS for subsequent retitration. Forexperiments utilizing modulator DNA, a 1:1 molar ratio of modulator DNAwas added to the primary DNA solution (DNA melting temperature is ˜55°C.).

Fluorometry and Titration. Fluorometric analysis was performed on aSafire dual monochromator multifunction microtiter plate reader (Tecan,Boston, Mass.). Samples were excited at 510 nm, and emission wasrecorded at 600 nm. A 5-μL aliquot of 2× solution containing dilutedexplosive was added to each well at room temperature for 5 minutes togive a final concentration in 100 μL. Each point of a titration wasperformed in triplicate (three separate wells), and FRET efficiency at600 nm was monitored during experiments. Titrations are plotted againstthe change (loss) in fluorescence compared to the 0 concentration point.In the figures, error bars appear where appropriate; if not, the erroris within the point value. Approximate binding constants K_(app) wereestimated from the second derivative of titration curves and rounded towhole integers.

A homogeneously labeled α-TNTscFv protein and sensor was desirable.Site-specific labeling of proteins is easily accomplished on uniquecysteine residues. The current α-TNTscFv protein already contains fourcysteines, which are required to form intrachain disulfide bonds tostabilize the variable heavy (VH) and variable light (VL) domains andhelp maintain the proteins' integrity and recognition function. Sincethe introduction of additional cysteines could result in detrimentaldisulfide bond formation during the expression and folding process and anonfunctional “disulfide scrambled” protein, an intuitive approach basedon modeling and design was undertaken. A model was constructed of theα-TNTscFv protein for the determination of optimum mutational sites.Selection criteria included surface-exposed residues to facilitatelabeling, proximity to peptide turns, location distal from the bindingsite, location distal from the already present cysteines, and selectionof residues, which, if changed, would have minimal affect on overallstructure. The four sites selected were Gln13, Leu145, Ala210, andAla241. Transformants were obtained for the first three mutationalsites. The Ala241 site is proximal to the

HISHISHISHISHISHISGLYGLYSERGLYGLYHISHISHISHISHISHIS (SEQ ID NO: 10)region and this may have interfered with mutagenic oligonucleotidehybridization. After DNA sequence verification of the three mutantsobtained, growth and induction revealed that only the strains expressingthe α-TNTscFv Ala210Cys and Leu145Cys mutants produced any proteinexpression. TNB binding was assayed by ELISA, and results indicted thatonly the α-TNTscFv-Leu145Cys mutant protein had any appreciable TNBbinding capacity. This mutant was subsequently dye-labeled with AFF 532and still retained its TNB binding capacity when reassayed using ELISA.

Sensor Assembly, Titration versus TNT, and Regeneration. The sensor wasassembled using the AFF 532-labeled α-TNTscFv-Leu145Cys protein.Attempts at tethering the dye-labeled TNB DNA arm and the labeledprotein separately resulted in low amounts of functional sensorassembly; thus, both elements were prebound prior to tethering to the NAsurface. This is in contrast to the maltose targeting prototype, whichwas assembled sequentially. By pre-binding prior to tethering, moreactive sensor sites consisting of both elements in functionalorientations were attached to the surface and available for regenerationafter washing. Following sensor self-assembly, TNT in solution wastitrated. The resulting loss of FRET-based fluorescence at 600 nm wasplotted against the concentration. TNT concentrations higher than 50mg/L were not used, as TNT solubility in aqueous solutions saturates atconcentrations approaching 100 mg/L. A lower limit of detection of 1mg/L TNT (1 part per million/ppm) was noted for this sensing assembly.The binding curve appears to be a relatively linear function over thisseries of concentrations tested. The sensor assembly was washed using 10volumes of 200 μL PBS and then regenerated in 50 μL of PBS for 24 h at4° C. A second TNT titration was performed, the sensor washed and thenregenerated for 1 hour at room temperature, and then a third titrationwas performed. Similar control experiments performed using sensorassemblies incorporating a dye-labeled DNA arm lacking the TNB moiety orsubstituting Cy3-labeled myoglobin for the α-TNTscFv resulted in nochange in fluorescence upon TNT addition. Additionally, the 600-nm TAMRAemission for these control assemblies was substantially lower, alsoindicative of inefficient FRET. These results confirm that the samesensor binding of the terminal DNA arm recognition analogue and sensorregeneration performance seen in the maltose sensing prototype ispresent in the current TNT sensing assembly.

Sensor Specificity and Modulation. To determine specificity andcross-reactivity, sensor assemblies were tested against a variety of TNTstructural analogues. Not surprisingly, TNB elicited the highest sensorresponse in terms of absolute fluorescence change, followed closely byTNT. This result can be attributed to the fact that the antibodyfragment was originally selected against TNB and the analogue attachedto the DNA arm is also TNB. Thus, TNB will be the most effectivecompetitor for binding sites. RDX and 2-A-4,6-DNT caused significantlyless response, with 2,4-DNT eliciting the least response. Since2-A-4,6-DNT elicits all of its response between 5 and 20 mg/L, theresult is almost a complete “classical” binding curve in comparison tothe results elicited from the other explosive compounds. It should benoted that, in this format, TNT cannot be distinguished from thestructurally related competitors until higher concentrations. Asmentioned, issues of solubility limit the concentration range that canbe tested for these compounds, limiting them to the “linear” portion ofthe curve. Nevertheless, these results very closely parallel thecompetition ELISA format used to test the precursor anti-TNB scFvselected. This demonstrates that the original α-TNTscFv specificity isretained even after multiple modifications including the following: (1)appending a (His)₆-spacer-(His)₆, (2) point mutation to a cysteine, (3)dye labeling of the cysteine, (4) surface tethering, and (5) interactingwith the dye-labeled TNB DNA arm as part of sensing. This also parallelsthe specificity retained by the maltose sensing prototype after similarmodifications.

The dynamic sensing range of the maltose sensing prototype could bemodulated by “stiffening” of the tether arm through the addition of aDNA complementary to the DNA linker. Although the binding constantessentially remained the same, the binding curve of the modulatedprototype became broader, significantly increasing the useful sensingrange. Similar experiments were performed with the current TNT sensingassembly. During self-assembly, modulator DNA was added to thehybridizable flexible DNA linker, significantly increasing the amount ofdouble-stranded DNA from 16 of 44 bases (˜35%) in the unmodulated sensorto 36/44 (˜80%) when modulated. The lower limit of TNT detection of themodulated sensor dropped ˜10-fold from 1 to 0.1 mg/mL (1 ppm to 100parts per billion/ppb). In parallel to the prototype, these results showthat the modulation mechanism can be exploited to adjust useful sensorproperties. The choice of C6 chain, 5′-amino-C6 and 15-carbon spacer inthe DNA arm was driven by availability for incorporation duringautomated DNA synthesis. It should be noted that adjusting the length ofthe single-stranded DNA arm (as opposed to rigidity) in the maltosesensing prototype did not effect binding properties. Thus, adjustingthese alkane moieties in terms of length were not considered critical inthe current sensor as well. “Switching in” of a more sensitive scFvantibody fragment would be another mechanism of adjusting sensorproperties.

The TNT sensing assembly demonstrated retains almost all of thedesirable properties of the MBP-based prototype while targeting adifferent analyte. Among the useful features of this sensor are the useof robust avidin-biotin technology for surface tethering and the ease ofself-assembly in a microtiter plate format, which also facilitatesanalysis by readily accessible fluorescent plate readers. The prototypeand current sensor use dye-based FRET; however, signal transduction maybe expanded to other proximity-sensitive methods such as electrochemicaldetection. The choices of FRET dyes and their locations on either moduleare also interchangeable, which can allow optimization to address anypotential distance requirements. Furthermore, the sensor can beregenerated for subsequent reuse. In these experiments, an upper limitof 6-8 regenerations was obtained. Regeneration in less than 1 hour isfeasible. Sensor response in the current microtiter well format islimited by technician handling time, which suggests that real-timesensing in a flow cell is feasible.

The modularity of the sensor design disclosed is demonstrated by readilyadapting it to target another analyte. As such, the sensitivity of thebiorecognition element was not a major criterion. However, this does notpreclude the current sensor from actual use as a solution-phase TNTsensor. More sensitive (His)₆-appended TNT recognition elements areavailable that demonstrate nanomolar sensitivity. The current α-TNTscFvfragment could undergo another round of evolution to select for higheraffinity mutants. Although the modulated behavior of the sensor wasmodest, 10-fold decrease in limit of detection, the conservation of thisfeature seen is important. The addition of DNA to stiffen the armcreates sensors that are more sensitive to perturbation, thus increasingsensitivity and yielding a lower limit of detection. Due to the issuesof TNT solubility, the same broadening of the dynamic sensing range withDNA stiffening could not be fully tested. However, this result suggeststhat modulation through careful control of arm stiffness/kinetics can befurther exploited to yield sensing assemblies with variable control overthe desired affinities. The combination of “switching in” of mutantproteins with different affinities and sensor arm modulation may alsopotentiate the sensitivity attainable.

The modular nature and adaptability of this sensor design can easily beadapted to target another analyte and yet remain functionally robust.Choices of biorecognition elements that can fit directly into thecurrent format include (His)₆-appended proteins such as bPBPs, scFvfragments, or even soluble fragments of cloned cellular receptors. The(His)₆ motif is commonly engineered into many cloned proteins for facilepurification over Ni-NTA media. Additionally, an analogue of the primaryanalyte of interest must be attached to the distal end of the DNA armand many covalent and noncovalent chemical linking strategies are knownin the art to address this. A variety of choices are available for boththe biorecognition module and the modular arm. Adaptation of this sensorfor testing in a drug discovery assay where a variety of compounds withdiffering affinities for the biorecognition module can be screened inparallel is possible. Multianalyte or “multiplex” sensing is anotherpossibility. Other applications may include providing a variety ofsensors in a microtiter well format for screening or quantitation ofmany different analytes such as in a clinical setting. Sensor assembliescan be dried, stored, and reconstituted for later use. This suggestedthat sensors could be assembled on microtiter plates and dried/storedfor later use in other locations. Although demonstrated in a microtiterwell plate format, assembly of these sensors in flow cells and on othersurfaces, including nanoparticle surfaces, is possible.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

1. A modular biosensor comprising: a biorecognition element, comprisinga biorecognition module, said biorecognition module being labeled with afirst signaling module; a modular arm element, comprising of a flexiblearm and a recognition module, wherein said modular arm element islabeled with a second signaling module, wherein said modular arm elementis specifically oriented in reference to the biorecognition element,wherein said biorecognition element is linked to said modular armelement, wherein said biorecognition module and said recognition moduleof said modular arm element bind, wherein said first and secondsignaling modules are in close proximity to each other to establish abaseline fluorescence resonance energy transfer (FRET); and a detectorfor detecting a change in FRET.
 2. The modular biosensor of claim 1,wherein said biorecognition element is linked to said modular armelement either covalently or non-covalently.
 3. The biosensor of claim1, wherein said signaling module is a dye, a quencher, anelectrochemically active group, a quantum dot or an enzyme.
 4. Thebiosensor of claim 1, wherein said biorecognition module is a protein,an enzyme, a receptor, a bacterial periplasmic binding protein, anantibody fragment, a peptide, an aptamer, a carbohydrate, DNA, PNA, RNA,or other macromolecule.
 5. The biosensor of claim 1, wherein saidbiorecognition module is at least one protein, an enzyme, a receptor, abacterial periplasmic binding protein, an antibody fragment, a peptide,an aptamer, a carbohydrate, DNA, PNA, RNA, or other macromolecule. 6.The biosensor of claim 1, wherein the flexible arm is comprised of aSS-DNA, a DS-DNA, a combination of a SS-DNA and aDS-DNA, a thiolatedDNA, a RNA, a thiolated RNA, a linear homopolymer, a linear copolymer, ablock copolymer, a PNA, an α peptides, a β peptides, a protein, apolymer, or an oligosaccharide.
 7. The biosensor of claim 1, wherein therecognition module is comprised of at least one antigen, epitope,analyte, substrate, protein, peptide, toxin, sugar, biological agent, oranalogs of said antigen, epitope, analyte, substrate, protein, peptide,toxin, sugar, or biological agent.
 8. The modular biosensor of claim 1,wherein said biorecognition element is attached to a surface by asurface attachment module by surface attachment.
 9. The biosensor ofclaim 8, wherein said surface attachment module is comprised of biotin,avidin, antibody, reactive thiol, reactive amine, non-reversible enzymesubstrate, protein A, protein G, protein L, DS-DNA, or PNA.
 10. Thebiosensor of claim 8, wherein said surface attachment is biotin-avidinchemistry, metal-affinity coordination, thiol bonding, hydrophobicinteractions, or DNA-directed immobilization.
 11. The biosensor of claim8, wherein said surface is comprised of a polystyrene micro-titer platewell, a solid planar macromaterial, a spherical or other shapedmaterial, a microscopic planar, spherical or other shaped micro- ornanomaterial, a nanocrystalline, a modified nanocrystalline material, amolecular or biomolecular assembly composed of protein, DNA, RNA, PNA,morpholino DNA, or other biomolecule and their derivatives, amolecularly templated material, a polymer, or a mineral.
 12. Thebiosensor of claim 1, wherein said biorecognition element and saidmodular arm element have a specific binding affinity.
 13. The biosensorof claim 12, wherein said specific binding affinity is modulated byaltering said biorecognition module.
 14. The biosensor of claim 12,wherein said specific binding affinity is modulated by temperature. 15.The biosensor of claim 12, wherein said specific binding affinity ismodulated by altering the properties of the flexible arm of the modulararm element.
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 31. A method of making a modularbiosensor, comprising the steps of: providing a biorecognition module;labeling the biorecognition module with a signaling module; synthesizinga modular arm element; labeling the modular arm element with a signalingmodule; linking the biorecognition module to the modular arm element;and binding the biorecognition module and the modular arm element byself assembly to establish a baseline FRET.
 32. A method of making amodular biosensor, comprising the steps of: providing a biorecognitionmodule; labeling the biorecognition module; synthesizing a modular armelement; labeling the modular arm element; attaching the biorecognitionmodule and the modular arm element to a surface; and binding thebiorecognition module and the modular arm element by self assembly toestablish a baseline FRET.
 33. A method of making a modular biosensor,comprising the steps of: providing a biorecognition module; labeling thebiorecognition module; synthesizing a modular arm element; labeling themodular arm element; binding the biorecognition module and the modulararm element by self assembly to establish a baseline FRET; and attachingthe biorecognition module and the modular arm element to a surface. 34.A biosensor array comprising a plurality of biosensors, wherein at leastone of the plurality of sensors is a sensor according to claim
 1. 35. Asensor array according to claim 34, wherein each of the sensors is asensor according to claim 1.